The nuclear lamina, a filamentous protein meshwork lining the nuclear envelope (NE), contains a polymer of lamins and associated transmembrane proteins of the inner nuclear membrane. The importance of the lamina in cellular functions is underscored by the findings that mutations in the genes for lamins A/C and lamina-associated transmembrane proteins cause over 15 inherited human disorders ("laminopathies"), including many dystrophies that affect heart and skeletal muscle. The long-term objectives of this project are to promote an understanding of the lamina's role in cell organization and functions, and its involvement in human diseases. Recently, 67 novel putative NE transmembrane proteins (NETs) have been identified in a comprehensive proteomics analysis of the NE. Expression profiling has revealed that 6 of these NETs are significantly up-regulated during myoblast differentiation and are expressed at high levels in adult skeletal muscle relative to other tissues, indicating an important role in muscle development. This proposal will focus on a 3 of these NETs, which have been validated to be authentic NE proteins, and which have putative roles in signaling based on their homologies to known proteins. The specific aims are to: 1) Carry out RNAi- mediated gene silencing in a myoblast differentiation model, and molecularly analyze the phenotypic defects that may arise related to differentiation, signaling responses related to muscle regulation, or NE structure. 2) Biochemically analyze specific NET functions suggested by their sequence homologies, and immunolocalize the endogenous NETs at the light and EM levels. 3) Implement MudPIT proteomic analysis of NET- containing protein complexes solubilized from cells to identify other NE proteins that interact with the NETs, and use in vitro binding studies to extend the results of the proteomics. This work is expected to provide a new level of insight on the NE as a signaling platform in the nucleus, and to promote an understanding of how mutations in NE and lamina proteins cause human diseases, particularly diseases affecting striated muscle.